www.criver.com EVERY STEP OF THE WAY 360 DIAGNOSTICS™ Serologic Methods Manual Table of Contents I. Overview ..................................................................................................................................................................... 2 II. Enzyme-Linked Immunosorbent Assay (ELISA) ........................................................................................................... 3 A. Methodology Overview.......................................................................................................................................... 3 B. Materials ............................................................................................................................................................... 3 C. Sample Preparation ............................................................................................................................................... 7 D. Testing .................................................................................................................................................................. 8 E. Results Interpretation: Charles River Scoring System........................................................................................... 10 F. Troubleshooting .................................................................................................................................................. 11 III. Indirect Fluorescent Antibody Test (IFA) ..................................................................................................................... 12 A. Methodology Overview........................................................................................................................................ 12 B. Materials ............................................................................................................................................................. 13 C. Sample Preparation, Collection and Storage ........................................................................................................ 14 D. Testing ................................................................................................................................................................ 15 E. Results Interpretation........................................................................................................................................... 16 F. Troubleshooting .................................................................................................................................................. 17 IV. Appendices ............................................................................................................................................................... 18 A. Equipment List for Serology Testing..................................................................................................................... 18 B. Reagent Suppliers for Serology Testing................................................................................................................ 18
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Serologic Methods Manual - Charles River Laboratories · Serologic Methods Manual 3 II. Enzyme-Linked Immunosorbent Assay (ELISA) A. Methodology Overview The indirect ELISA method
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www.criver.com
EVERY STEP OF THE WAY
360 DIAGNOSTIC S™
Serologic Methods ManualTable of ContentsI. Overview ..................................................................................................................................................................... 2
II. Enzyme-Linked Immunosorbent Assay (ELISA) ........................................................................................................... 3
A. Methodology Overview .......................................................................................................................................... 3
B. Materials ............................................................................................................................................................... 3
C. Sample Preparation ............................................................................................................................................... 7
D. Testing .................................................................................................................................................................. 8
E. Results Interpretation: Charles River Scoring System ........................................................................................... 10
F. Troubleshooting .................................................................................................................................................. 11
III. Indirect Fluorescent Antibody Test (IFA) ..................................................................................................................... 12
A. Methodology Overview ........................................................................................................................................ 12
B. Materials ............................................................................................................................................................. 13
C. Sample Preparation, Collection and Storage ........................................................................................................ 14
D. Testing ................................................................................................................................................................ 15
E. Results Interpretation........................................................................................................................................... 16
F. Troubleshooting .................................................................................................................................................. 17
IV. Appendices ............................................................................................................................................................... 18
A. Equipment List for Serology Testing ..................................................................................................................... 18
B. Reagent Suppliers for Serology Testing ................................................................................................................ 18
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I. OverviewAt Charles River Laboratories, ensuring the quality of animal models used in biomedical research is our highest priority. To
accomplish this goal, we have developed a number of diagnostic testing strategies and methods to determine if animals have
been exposed to adventitious infectious agents. Infections of immunocompetent animals are generally transient, yet serum
antibody responses to infection often can be detected within days to weeks and persist throughout the life of the host. As
immunoassays for antibodies to etiologic agents are rapid, inexpensive, specific and sensitive, serology is the primary diagnostic
methodology by which laboratory animals are monitored for adventitious infections with viruses, mycoplasma, and other
fastidious microorganisms. While serologic tests are designed to be sensitive and specific, false-positive and -negative results
do occur (Figure 1). We strongly recommend that clients confirm new positive findings by both alternative diagnostic methods
and testing additional animals. The enzyme-linked immunosorbent assay (ELISA) and indirect fluorescent antibody test (IFA) are
extremely sensitive. In the case of the ELISA, the test procedure and reading of results are amenable to automation.
The purpose of this manual is to outline the ELISA and IFA procedures used at Charles River. The following topics will be covered
for each assay:
The information provided is intended to help our clients develop a rodent serologic testing program. We encourage clients to
contact us with any comments or questions regarding this manual.
Figure 1
Serologic Methods Manual
• Methodology overview
• Materials/reagent preparation
• Sample preparation
• Step-by-step assay procedure
• Results interpretation
• Troubleshooting
3Serologic Methods Manual
II. Enzyme-Linked Immunosorbent Assay (ELISA)
A. Methodology Overview
The indirect ELISA method is one of the assays most often used for detection of antibodies. Typically, microbial-specific
antigen is immobilized on the surface of wells in microtiter plates made of specially-prepared polystyrene. Serum samples
are incubated in the well, to which antibodies may bind. Unbound antibodies are removed by washing. Antibodies that have
bound to the attached antigen are demonstrated by incubating first with an enzyme-conjugated anti-immunoglobulin, and
then, following a wash to remove unbound conjugate, with a chromogenic enzyme substrate. A colored product develops at
a rate proportional to the amount of antigen-specific antibody in the specimen. Color intensity can be assessed visually or
spectrophotometrically (in absorbance units) with an ELISA reader.
Ideally, antibodies bind specifically to their corresponding antigen. In practice, however, they may bind nonspecifically
to antigens. In addition to the antigen-coated well, we incubate each sample in an adjacent tissue control well to detect
nonspecific binding. The tissue control does not contain any material from an infectious agent but is usually prepared from the
host system in which the infectious agent is propagated. For example, we propagate MHV virus in mammalian NCTC cells.
The tissue control for MHV is a cellular lysate of uninfected NCTC cells. In the case of M. pulmonis, however, the tissue control
is another cross-reacting rodent mycoplasma, M. arthritidis. Wild-type baculovirus-infected insect cell lysate is used as the
tissue control for recombinant antigens (e.g., NS-1, Hantaan).
A positive result is recorded for a sample if color develops in the antigen well, but not in the tissue control well. Little or no
color development in either the antigen or control well for the sample is recorded as a negative result. When a color reaction
occurs in the tissue control well in addition to the antigen well, the result is recorded as a tissue-control reaction (TC). A TC
result is considered nonspecific and does not indicate whether a sample is antibody-positive or -negative. In our Serology
Department, we usually retest the sample by an alternative method and, if necessary, we test additional samples from the
same source. A more detailed explanation of score calculation and interpretation is in Section II-E.
B. Materials
1. Equipment
a) Incubator at 35-40 °C
For the best sensitivity and reproducibility, test plates should reach the incubation temperature as quickly as possible. To
accomplish this, we recommend using a mechanically-circulated hot air incubator rather than a convection incubator.
Make sure to use an incubator with adequate shelf space to avoid stacking plates more than two high. We also
recommend covering the plates with sealing tape and incubating them in a humidified chamber to prevent evaporation;
we use a covered plastic tub with water-dampened paper towels.
b) Plate washer (optional but recommended)
Washing to remove unbound antibody is a crucial step in any solid-phase immunoassay. Although washing can be
adequately performed without specialized equipment, a programmable 96-well plate washer offers several important
advantages. These include consistency, speed and containment of potentially contaminated fluids aspirated from test
plate wells.
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c) Reader (optional but recommended)
While test results can be read visually, we recommend using an ELISA plate reader equipped with a light source and filter
appropriate to the color produced by the substrate. We strongly recommend the use of Kirkegaard & Perry Laboratories’
(KPL) 1-component substrate ABTS-H2O2 that requires a 405 nm filter. Most ELISA plate readers have at least one RS-
232 serial port, which allows them to send results to a computer for analysis. We have found that using a computer to
receive absorbance values and compile reports saves time and effort, and prevents errors.
d) Pipettors
Reagent and sample preparation and sample transfers require various pipettes that accurately dispense volumes of 10 to
1,000 µL. For most purposes, the following pipettes are adequate:
Type Volume (µL)
Single-channel, adjustable volume
2-10
10-100
100-1,000
8- or 12-channel, adjustable volume5-50
50-300
Repeating pipette with 8- or 12-channel 50
2. Description of key reagents
a) Antigen- and tissue-control-coated test plates
The pattern used to coat our ELISA plates is shown in Figure 2. Wells in rows A, C, E and G contain attached test-plate-
specific antigen. The remaining wells (rows B, D, F and H) are coated with the corresponding tissue control. We routinely
add the ELISA assay controls into column 12.
Our test plates (intended for research use only) are available in a 96-well format with removable 8-well strips. Plates
with removable strips offer the flexibility of doing fewer than 48 tests at a time. For a list of available ELISA plates, go to
A result with a high tissue score can still be interpreted as negative, provided that the antigen score is less than 2.5. A result is
considered nonspecific and recorded as TC when both ScoreAG and ScoreTC are > 2.
We strongly recommend that you confirm new ELISA positive findings by alternative diagnostic methods (IFA and/or Western
blot), alternate methodologies (PCR), and by testing the same and/or additional animals. Alternatively, suspect serum samples
can be shipped to Charles River Serology for confirmation testing.
Serologic Methods Manual
11Serologic Methods Manual
F. Troubleshooting
The following table describes common ELISA issues and their probable cause
Observation
Possible Cause
Component Problem
No color in high and low positive control wells
Control sera
• Not added• Incorrect specificity• Diluted improperly (i.e., too dilute)• Inactivated by improper storage or repeated freeze-thaws
Conjugate• Incorrect specificity• Too dilute• Inactivated by improper storage or repeated freeze-thaws
Substrate • Alternate ABTS substrate used other than recommended KPL catalog #50-66-06
Antigen• Too dilute or of low potency• Incorrect agent• Degraded due to improper storage
Reader• At wrong wavelength• Bulb burned out• Out of calibration
Weak color development in high and low positive control wells
Components listed above
• Plate read in wrong orientation• Problems listed above
Incubation• Temperature too low or exceedingly high• Time too short
Excessive background
Washer• Too few fill-aspirate cycles• Wash incompletely aspirated after each fill• Fill volume low
Serum sample
• Sticky due to improper collection and storage or bacterial contamination• Dilution too low or not diluted in BLOTTO serum diluent• From animal immunized or used to grow tumor cells; animal with autoimmune
disease• Older-age animal used
Conjugate• Dilution too low• Poor quality (try another lot or different vendor)
Substrate • Activated nonspecifically prior to being added to plate or by contaminants in plate
Antigen• Binds antibody in serum or conjugate nonspecifically because “sticky” or used at
too low a dilution
Incubation
• Time too long• Temperature too high• Plates were not completely covered to prevent evaporation of well contents• No humidity to prevent evaporation of well contents
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III. Indirect Fluorescent Antibody Test (IFA)
A. Methodology Overview
The steps of the IFA are very similar to those of the ELISA. Virus-infected cells and uninfected cells are fixed to wells on a
glass slide. The fixative is usually cold acetone, which permeabilizes the cell membrane, making the intracellular viral antigens
more accessible to antibodies. As with the ELISA, unbound antibodies are removed by washing. Instead of the enzyme
conjugate and substrate indicator system used in the ELISA, the binding of primary antibodies to the slide wells in the IFA is
demonstrated with a fluorescent dye-conjugated anti-immunoglobulin. After washing to remove unbound conjugate, slides
are covered with buffered glycerol and examined with a fluorescence microscope. Epi-illumination is recommended due
to the fluorescence being much brighter than transmitted-light darkfield fluorescence, resulting in a clearer, crisper image.
Fluorescence microscopes have a light source with an exciter filter to exclude all but the appropriate wavelengths and a
reflector/barrier filter combination to reflect the light onto the slide, so fluorescence may be observed.
In the IFA, the morphology and location of fluorescence can be evaluated to differentiate specific from nonspecific reactions.
This is not true for most other serologic tests and is a major advantage of the IFA. Bright, granular fluorescence is typical of
a specific antibody-viral antigen reaction. By contrast, diffuse fluorescence suggests a nonspecific reaction. Fluorescence
may be mostly nuclear or cytoplasmic, depending on the virus (see table below). Nuclear fluorescence is characteristic
of DNA viruses (e.g., the rodent parvoviruses MVM, MPV, RPV, KRV, RMV and H-1). However some minimal cytoplasmic